Background: Foxp3+CD4+CD25+ regulatory T cells (Treg)-mediated immune suppression is crucial for immune evasion by tumor cells and an obstacle for successful tumor immunotherapy. Hence, the ability to disrupt Treg function is of major therapeutic significance. Although Foxp3 is a master regulator of Treg, Foxp3 expression is not sufficient to account for the suppressive capacity of Tregs. It has been suggested that Foxp3 needs to associate with other co-factors in order to suppress non-Treg (T effector) genes and enforce Treg associated gene expression and function. Recently, we found that Yes-associated protein (YAP), a downstream co-activator of the Hippo pathway, is highly expressed by Tregs and is critical for Foxp3-mediated suppressive activity. Furthermore, T cell specific YAP knockout mice mount superior immune responses to implanted B16 melanomas. We hypothesize that YAP is an attractive target for immunotherapeutic strategies aimed at breaking tolerance and enhancing anti-tumor immunity in the cancer setting. Specific Aims: In the current proposal, we are seeking to: 1) Further dissect the molecular mechanisms by which YAP facilitates Foxp3+ Treg function; 2) Understand the consequences of YAP deletion for Treg cell differentiation and function; and 3) Explore the anti-tumor efficacy of YAP inhibitor(s) alone or in combination with immune checkpoint blockade. Objectives & Significance: These studies will expand our understanding of the mechanisms behind YAP facilitates Foxp3 regulation and Treg function. In so doing we will further dissect the molecular mechanism by which YAP facilitates Foxp3-mediated Treg function. Furthermore, we will explore Yap as a potential novel therapeutic target by pharmacologically manipulating YAP activity, and testing various inhibitors for efficacy as breakers of immune tolerance, which is a major obstacle for anti-cancer immunotherapy. The use of such Yap inhibitors in combination with other immune modulators such as anti-PD-1 is expected to improve the effectiveness of immunotherapy, and boost anti-tumor immunity and patient survival. Methodology: In these studies we will deploy biochemical, molecular biology, genetic and bioinformatic approaches to further dissect the role of YAP in Treg cell biology, and attempt to discover known and novel inhibitors of Yap that are effective alone and synergistic with immunotherapies in reducing tumor burden. Both novel and known YAP inhibitors will be tested for their capacity to undermine Treg function and break immune tolerance in vitro and in vivo. Expected Results & Implications: Our experiments will reveal a novel role of YAP in Treg cell function. We predict that a detailed understanding of the physiological role of YAP induction and its impact on Foxp3 and Treg function will provide insight into therapeutic targeting of this pathway. The use of YAP inhibitors in combination with proven checkpoint targeting agents may yield even better anti-tumor efficacy.